Method and device for statistical tissue sampling using microdevices

ABSTRACT

The present invention utilizes tetherless microtools to biopsy tissue. The invention provides a device and method for deployment and retrieval of tetherless microtools. The size of the microtools ensures that tissue damage at a site targeted for biopsy is negligible. As such, large numbers of microtools may be deployed ensuring that a true statistical sampling of biologic tissue is performed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 USC §371 National Stage application ofInternational Application No. PCT/US2012/036386 filed May 3, 2012, nowpending; which claims the benefit under 35 USC §119(e) to U.S.Application Ser. No. 61/541,962 filed Sep. 30, 2011, U.S. ApplicationSer. No. 61/525,364 filed Aug. 19, 2011 and U.S. Application Ser. No.61/483,536 filed May 6, 2011. The disclosure of each of the priorapplications is considered part of and is incorporated by reference inthe disclosure of this application.

GRANT INFORMATION

This invention was made with government support under Grant No. DP2-OD004346-01 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates generally to diagnostics and morespecifically to a method and device for obtaining randomized tissuesamples for statistical sampling and analysis for diagnostic andprognostic evaluations.

Background Information

Over the past several years, numerous genetic and epigenetic alterationswere identified in a wide variety of disorders. In cancer, inparticular, multiple alterations of etiologic and/or diagnostic valueshave been identified. Gene expression studies have identified singlegenes, or panels of genes to accurately diagnose normal, pre-malignantor malignant lesions, as well as to differentiate between subtypes ofcancer. In addition to mRNA species, microRNA (miRNA) alterations havebeen determined to be associated with various inflammatory or malignantconditions, and can be used for diagnostic purposes. Epigeneticalterations have also been shown to be predictive of malignanttransformation.

The diagnostic accuracy of various tests based on genetic or epigeneticalterations in particular, and histological analysis in general, is,however, intrinsically linked to the quality and volume of tissue thatis available for analysis. As such, obtaining the relevant tissue isessential for the diagnosis.

Well executed tissue sampling procedures are especially crucial forbiopsy of potential mucosal abnormalities in the gastrointestinal (GI)tract and elsewhere in the body that cannot be readily diagnosed basedon their appearance on visual observation; e.g., during endoscopicexamination. For example, several patchy conditions of the stomach,including infection with Helicobacter Pylori, autoimmune atrophicgastritis and dysplasia, mandate obtaining biopsies at several locationsin the stomach. Similarly, biopsy-based diagnosis of microscopiccolitis, dysplasia in ulcerative colitis, and others mandate obtainingmultiple biopsies.

With the recent advent of endoscopy and laparoscopy, access to theinside of the human body has become routine. However, visual inspectionis continually relied upon to aid diagnosis by obtaining tissue biopsywith tethered and relatively large biopsy forceps devices. Spacelimitations, time limitations and tissue damage limitations allcontribute to dismal ability of performing a true statistical samplingof biologic tissue.

A key step to increase the number of biopsy sites while preventingunnecessary tissue damage is the miniaturization of the biopsy tools.Smaller tools are being developed for use at different biopsy sites tominimize tissue loss. However, they remain limited in application to theconfined, low visibility environment of bodily passages.

SUMMARY OF THE INVENTION

The present invention provides a clinically appropriate method forbiopsying tissue in difficult to access regions of a human or animalbody. According to the method, a plurality of tetherless microtools isdeployed to tissue targeted for biopsy. A clinically useful method forretrieval of the microtools with the tissue samples is also provided.

The nanoscale size of microtools suitable for use in the inventionensures that tissue damage at the deployment site is negligible. Assuch, large numbers of microtools may be deployed in numbers sufficientto enable statistically significant sampling of the targeted tissue. Ina preferred embodiment, the microtools are adapted to open and closearound targeted tissue in response to specific stimuli such astemperature, allowing them to be used without need for additional biopsyinstruments.

In another aspect, the present invention provides a method fordeployment of a tetherless microtool into a cavity of a subject. Themethod includes: a) introducing a device comprising one or more magnetsinto a cavity of a subject; and b) deploying a tetherless microtoolmagnetically detachable from the one or more magnets to a tissue of thecavity.

In one aspect, the present invention provides a method of tissuesampling. The method includes: a) contacting a plurality of tetherlessmicrotools with a tissue of a subject, each microtool having a firstconfiguration; b) allowing the plurality of tetherless microtools toalter from the first configuration to a second configuration while incontact with the tissue, the second configuration adapted such that eachmicrotool grasps the tissue at a discrete location; and, c) retrievingthe plurality of tetherless microtools from the subject using a devicecomprising one or more magnets, wherein each microtool retains anindividual sample of the tissue upon retrieval, thereby performingtissue sampling.

In another aspect, the present invention provides a method for retrievalof a tetherless microtool from a subject. The method includes: Themethod includes: a) introducing a device comprising one or more magnetsinto a bodily cavity of the subject, the cavity defining or beingadjacent to tissue having at least one tetherless microtool attachedthereto, wherein the microtool is comprised in whole or in part of amagnetically responsive material; b) allowing the one or more magnets ofthe device to contact the tetherless microtool; and c) retrieving thetetherless microtool from the subject via magnetism by removing thedevice from the cavity, wherein the microtool retains a tissue samplefrom the cavity upon retrieval.

In yet another aspect, the present invention provides a method ofobtaining a randomized tissue sample during a diagnostic biopsy of asubject using tetherless microtools and the device of the presentinvention. The method includes: a) introducing a plurality of tetherlessmicrotools into a cavity of a subject; b) introducing a devicecomprising an expandable element having one or more magnets into thecavity; c) anchoring the device via the expandable element so as tofacilitate contact between the expandable element and the plurality oftetherless microtools; d) withdrawing the device so that the pluralityof tetherless microtools collect on the expandable element via magnetismand are removed from the subject, wherein each of the plurality oftetherless microtools comprise an individual tissue sample from thecavity; and e) processing the samples for diagnostic or prognosticbiopsy.

In another aspect, the present invention provides a device fordeployment or retrieval of a tetherless microtool from a subject. Thedevice includes one or more magnets configured for retrieval ordeployment of a magnetically responsive tetherless microtool. A systemfor biopsy use including the device further including a tetherlessmicrotool magnetically detachable to the one or more magnets.

In various embodiments, the tetherless microtools are deployed and/orretrieved from the subject using the device of the present invention. Incertain such embodiments, the device is configured as a catheter havingone or more openings in the elongated shaft of the device for deploymentof the plurality of tetherless microtools. In some embodiments, theshaft of the device further includes a main lumen extending the entirelength of the shaft which is configured to allow passage therethrough ofan endoscope or a catheter.

In some embodiments, the device further comprises an expandable element.In certain of these embodiments, the expandable element is an inflatableballoon, resilient coil, stent or spring. If a balloon, device may beconfigured as a balloon catheter. In some embodiments thereof, magnetsare disposed on the expandable element, and may be integrated therein(e.g., within or as the material forming a coil, stent or spring) orprovided thereon as dots or strips (e.g., on the outer surface of aballoon). In some embodiments, the expandable element is disposed at thedistal end of the catheter shaft to provide ready contact between themagnets and deployed microtools.

In alternative embodiments, the tetherless microtools are constructed inwhole or in part of bioresorbable materials. As such, if left behind inthe body (whether intentionally or inadvertently), they will eventuallybecome absorbed.

In various embodiments, the method further includes analyzing theindividual samples retrieved from the tetherless microtools. Forexample, the analysis may include analysis of one or more nucleic acidsequences, e.g., genetic analysis, to diagnose a disease or condition.

In another aspect, the present invention provides a method of diagnosinga condition or disease in a subject. The method includes: a) obtaining atissue sample from a subject via the method of the invention; b)analyzing the tissue sample; and c) providing a diagnosis of thecondition or disease based on the analysis.

In another aspect, the present invention provides a method of providinga prognosis for a condition or disease in a subject. The methodincludes: a) obtaining a tissue sample from a subject via the method ofthe invention; b) analyzing the tissue sample; and c) providing aprognosis of the condition or disease based on the analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a-1h provide a series of schematic representations depictingfabrication and operation of a microtool, configured as a microgripper,in one embodiment of the invention. FIG. 1a shows use of a thin Cu layeras the sacrificial layer. FIG. 1b shows a pre-stressed Cr—Au bilayerpatterned and evaporated. FIG. 1c shows electroplating of ferromagneticNi as the rigid segments between the hinges and covered with Au. FIG. 1dshows patterning of thermo-sensitive polymeric trigger. FIG. 1e depictslifting-off the microgrippers from the substrate by dissolving thesacrificial layer. Figure if depicts closed microgrippers(conformational change to a second structural conformation) when exposedto transition temperature. FIG. 1g depicts optical microscopy imaging ofthe microgrippers on the Si substrate before the lift-off process. FIG.1h depicts optical microscopy images of different size microgrippersafter the lift off. The scale bars represent 400 μm.

FIG. 2a-2i provide images showing capture and retrieval of cells usingmicrogrippers depicted in FIG. 1 in one embodiment of the invention. Thescale bar is 1 mm long. FIGS. 2f and 2g are images showing microgripperscapturing cells. The scale bars are 100 FIGS. 2h and 2i are images of anex-vivo biopsy procedure showing microgrippers in one embodiment of theinvention is the intrahepatic bile ducts. The scale bar is 1 mm.

FIG. 3a-3c provide images of microgrippers patterned with bio-polymersin one embodiment of the invention. FIGS. 3d-3f are images showingconformational change of the microgrippers patterned with bio-polymersupon enzymatic degradation of biopolymer layers and closing/re-openingof the devices. FIG. 3g is a graphical plot showing the kinetics ofmicrogrippers patterned with bio-polymers closing upon exposure todifferent enzymes. The graphs plot experimentally measured diameterreduction ratio versus time on exposure to a variety of enzymes. Theline denotes the average value measured over 5 trials and shaded regiondenotes the standard deviation.

FIG. 4 is a series of pictorial representations of chemically actuatedmicrogrippers that are responsive to pH.

FIG. 5 is a schematic representation of one the device and methodologyin one embodiment of the invention.

FIG. 6 is a schematic representation of one the device and methodologyin one embodiment of the invention.

FIG. 7 is a schematic representation of one the device and methodologyin one embodiment of the invention.

FIG. 8a-8b provides pictorial representations of an ex-vivo colonoscopyperformed on porcine colon. FIG. 8a is an optical image of colonpreparation for endoscopic visualization. FIG. 8b is an endoscopic imageof porcine colon.

FIG. 9a-9d provides pictorial representations of deployment andretrieval of the microgrippers in an ex-vivo porcine model in oneembodiment of the invention. FIG. 9a is an image showing microgrippersdeployed in one area in the colon. FIG. 9b is an image showingmicrogrippers covering the colon surface, in a different deployment.FIG. 9c is a close up image of microgrippers closing on the colon wall.FIG. 9d is an image showing magnetic retrieval of the microgrippers.

FIG. 10a-10d provides pictorial representations including opticalmicroscopy images and gel electrophoresis results of tissue obtainedusing microgrippers. FIG. 10a is an image showing retrieved grippers ona magnetic retrieval device configured as a catheter in one embodimentof the invention. FIG. 10b is an image showing the retrieved tissueafter staining with trypan blue. Scale bars represent 200 μm. FIG. 10cis a image of an electrophoresis gel of PCR of cDNA from the tissueobtained with microgrippers (G) compared to negative control (N). FIG.10d is an image of an electrophoresis gel of PCR of genomic DNA fromtissue retrieved by grippers.

FIG. 11 is a pictorial representation showing catheter insertion in bileduct tissue sampling.

FIG. 12a-12b provides pictorial representations of optical microscopyimages. FIG. 12a is an image showing retrieved grippers on a magneticretrieval device configured as a catheter in one embodiment of theinvention. FIG. 12b is an image showing the retrieved tissue afterstaining with trypan blue. Scale bars represent 150 μm.

FIG. 13a-13i provide schematic representations depicting fabrication andoperation of a microgripper in one embodiment of the invention that canclose and re-open on exposure to enzymes. FIGS. 13a-13c are opticalimages of the grippers in the flat, closed, and re-opened statesrespectively. The arrow indicates the second set of hinges. Scale barsrepresent 200 μm. FIGS. 13d and 13f are schematic representations of themicrogrippers in the three corresponding states above. In FIG. 13d thegripper is held flat by the thick, crosslinked biopolymer (hatching).When the biopolymer is selectively degraded by enzyme 1 the modulusdecreases and the gripper closes. In FIG. 13f , the second trigger, arigid biopolymer insensitive to enzyme 1, is preventing a second set ofhinges from actuating, keeping the gripper closed. Subsequently thistrigger can be actuated by enzyme 2 to re-open the gripper. FIGS.13g-13i are cross sectional views of a magnified single hingeillustrating behavior of biopolymer trigger. FIG. 13g shows that bothbiopolymer layers are stiff, preventing all bending. FIG. 13g shows thaton degrading the biopolymer with enzyme 1, the second hinge remainsflat. FIG. 13i shows that when the second polymer is degraded by enzyme2 the hinge bends in the opposite direction.

FIG. 14 is a graphical representation depicting mathematical modeling ofthe actuation of microgrippers (of FIG. 13) as a function of moduli ofbiopolymers. Letter (a) of the Figure indicates that in the initialstate both polymers are stiff (moduli higher than 10⁴ Pa) and the entiregripper is flat. Letter (b) of the Figure indicates that when the firstbiopolymer is degraded and its modulus decreased to approximately 100Pa, the first set of six hinges bends causing the gripper to close.Letter (c) of the Figure indices that the remaining two hinges bend whenthe modulus of the second biopolymer trigger is decreased.

DETAILED DESCRIPTION OF TILE INVENTION

The present invention provides a method and device for performing tissuesampling utilizing a plurality of microdevices or microtools in atetherless fashion. As discussed herein, microtools useful in theinvention are relatively non-invasive and non-toxic devices. Accordingto the invention, microtools are deployed in numbers sufficient togreatly improve statistical sampling of biologic tissue and retrieved toperform a molecular and/or histological diagnostic assay of enhancedsensitivity compared to analysis of conventionally obtained biopsysamples.

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to particularcompositions, methods, and experimental conditions described, as suchcompositions, methods, and conditions may vary. It is also to beunderstood that the terminology used herein is for purposes ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyin the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described.

The present invention provides an innovative biopsying technique thatprovides significantly improved tissue sampling. The invention iscentered on the development of tetherless microtools, or microgrippers,that are deployed to a tissue via an invasive scope, such as anendoscope or laparoscope. After deployment, the microtools are activatedintraluminally to capture a plurality of tissue samples. The microtools,with tissue samples joined thereto, are then retrieved for analysisusing a retrieval device as described herein.

As used herein, the term “microtool” is used synonymously with the terms“microgripper” or “gripper” and is intended to refer to a device havingmultiple layers configured to allow for the structural configuration ofthe device to change from in a first configuration to a secondconfiguration in response to a stimulus, such as a change in a localenvironmental condition. Change from the first configuration to thesecond configuration allows for the device to grasp tissue via releaseof stored torsional energy in one or more layers of the device.

More specifically, microgrippers for use with the present invention aregenerally lithographically structured devices having an actuation layerand a control layer operatively connected to the actuation layer. Theactuation layer includes a stress layer and a neutral layer that isconstructed of materials and with a structure such that it storestorsional energy upon being constructed. The control layer isconstructed to maintain the actuation layer substantially in a firstconfiguration in a local environmental condition and is responsive to achange in the local environmental condition such that it permits arelease of stored torsional energy to cause a change in a structuralconfiguration of the lithographically structured device to a secondconfiguration, the control layer thereby providing a trigger mechanism.

Preferred microgrippers for use with the present invention are disclosedin International Publication No. WO 2009/111737, the entire contents ofwhich are incorporated herein by reference. Additional microgrippers foruse with the present invention are disclosed in Bassik et al. (J Am ChemSoc 132:16314-7 (2010)), Leong et al. (Proc Natl Acad Sci USA 106:703-8(2009)) and Randhawa et al. (J Am Chem Soc 130:17238-9 (2008)), theentire contents of which are also incorporated herein by reference.

In general, such microgrippers preferably mimic biological appendages,such as claws, with flexible joints between rigid regions. However,those of ordinary skill in the art will appreciate that microgrippersmay be any shape that may facilitate grasping of tissue. FIGS. 1-4,depict various shapes of microgrippers, such as generally star shapeddevices (FIGS. 1-3) and clam shaped devices (FIG. 4).

Microtools or microgrippers for use with the present invention mostpreferably include a material that is responsive to a magnetic field,such as a magnetic film, coating or solid deposit. Inclusion of amagnetic material in the microgripper allows them to be guided intoplace with an external magnetic field, and retrieved in turn. Use of amagnetic field allows a metallic microgripper to be magneticallydetachable with the device to facilitate removal or deployment of themicrogrippers. A variety of different magnets may be utilized. Forexample, the magnets may be permanent or non-permanent. Exemplarymagnets include, rare-earth magnets or magnets including lanthanideelements. Additional magnets include electromagnets which allow controlof the magnetic field by the user of the device to enhance manipulationof the microtool.

In one convenient form, the magnetic portion of the microgripper may becomprised of a ferromagnetic material. Non-limiting examples offerromagnetic materials include Co, Fe, Fe₂O₃, FeOFe₂O₃, NiOFe₂O₃,CuOFe₂O₃, MgOFe₂O₃, MnBi, Ni, MnSb, MnOFe₂O₃, Y₃Fe₅O₁₂, CrO₂, MnAs, Gd,Dy and EuO. The magnetic material may be incorporated into themicrogrippers in any manner that does not inhibit the device fromaltering conformation to allow grasping of tissue; for example, alongrigid, non-articulating portions of the microgripper body.

The microgrippers are bio-inert, and may also be wholly or partiallybioresorbable in construction. For example, the microgrippers may becomposed completely of bio-inert materials or may be coated withbio-inert materials. As number of materials useful as coatings are wellknown in the art. Examples of bio-inert coating materials includepolymers, such as polyimide, PEEK, polytetrafluorethylene,polyvinylidene fluoride, and polyamide, as well as certain metals, suchas gold.

The energy required for the gripping action is intrinsically provided tothe microgrippers as a consequence of residual stress stored in thejoints and can be released when the hinges are softened (especially ifpolymeric) or otherwise loosened by heating, delaminated ordisintegrated. As a result, the microgrippers require no wires, tethersor batteries to effectuate gripping. The microgrippers may be configuredto be responsive to a number of stimuli. In various embodiments, themicrogrippers are configured to be responsive to temperature, presenceof a chemical, enzymatic degradation, pH, a biomolecules, and the like.

FIGS. 1-4 show schematic representations of various microgrippers andtheir operation. FIGS. 1-2 show an embodiment of the invention in whichthe microgrippers are thermo-actuated sub-millimeter microgrippers asdisclosed in Leong et al. (Proc Natl Acad Sci USA 106:703-8 (2009)).FIG. 3 shows an embodiment of the invention in which the microgrippersare enzymatically actuated as disclosed in Bassik et al. (J Am Chem Soc132:16314-7 (2010)). FIG. 4 shows an embodiment of the invention inwhich the microgrippers are chemically actuated by pH.

Microgrippers of the present invention may be of a variety of sizes. Invarious embodiments, the microgrippers have a major dimension less thanabout 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mm. In an exemplary embodiment,the major dimension is less than about 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4,0.2 or 0.1 mm. Due to their small mm or sub-mm size, hundreds can bedispersed in a small amount of liquid and moved by fluid flow.

As discussed further herein and exemplified in the examples,microgrippers as disclosed herein can retrieve tissue samples, which canthen be successfully used for biological analysis, such as histologicalanalysis or evaluation of nucleic acid derived from the samples. Forexample, as illustrated herein, RNA and DNA extraction may be performedfollowed by polymerase-chain reaction (PCR) for several genes fromtissue retrieved by microtools. Since genetic and epigenetic diagnosesof cancer, inflammatory as well as other mucosal conditions are based onusage of RNA and/or DNA, the biopsy technique of the present inventioncan produce biologic material that is usable for such analyses.

There are several advantageous features of the microgrippers which allowstatistical tissue sampling for applications such as analysis of geneticmaterial contained in the samples. For example, the devices can befabricated and actuated en-masse in a cost-effective and reliablemanner. Also, the devices close in response to a change in localenvironment, such as body temperature, typically within 5 minutes whichenables actuation in a wireless or tetherless manner. Additionally, thedevices are relatively bio-inert to provide reduced risk if left behind.Further, the devices are small enough to be deployed with standardsurgical catheters. Finally, the devices possess magnetic elementsallowing magnetic retrieval and in situ tracking.

A device for deployment and/or retrieval of a tetherless microtool froma subject is also provided by the invention. The device includes one ormore magnets configured for retrieval or deployment of a magneticallyresponsive tetherless microtool. A system for biopsy use including thedevice comprises both the device, a tetherless microtool magneticallydetachable to the one or more magnets and, optionally, a deliveryinstrument (e.g., an endoscope, laparoscope or other invasive scope).

In particular, one or more magnets constructed as described with respectto the microgrippers are also disposed on or within the microgripperdeployment/retrieval device according to the invention. The magnets maybe detachably joined to the corresponding magnetically responsivematerial on the microgrippers.

One of skill in the art would understand that such magnets may beincorporated into the device in a number of ways. For example, magnetsmay be disposed on or within the device. In various embodiments, amagnet may be integrated into the device during manufacture or disposedon the surface of the device. One of skill in the art would alsoappreciate that any number and size of magnets may be utilized as wellas being incorporated into the device in any pattern that facilitatesdeployment or retrieval of a microgripper. The magnetic configuration islimited only by the shape of the device.

To provide a surface for carrying the retrieval magnets, the device mayfurther comprise an expandable element, such as an inflatable balloon,resilient coil, stent or spring. If a balloon, the device may beconfigured as a balloon catheter. In some embodiments thereof, magnetsare disposed on the expandable element, and may be integrated therein(e.g., within or as the material forming a coil, stent or spring) orprovided thereon as dots or strips (e.g., on the outer surface of aballoon). In some embodiments, the expandable element is disposed at thedistal end of the catheter shaft to provide ready contact between themagnets and deployed microtools. Those of ordinary skill in the art willbe readily familiar with the myriad of such expandable element devicesthat are commercially available and suitable for, or adaptable to, usein the invention.

In various embodiments, especially those utilizing a balloon as anexpandable element, the device may further include a radiopaque materialto allow for detection of the position of the device. A number ofradiopaque materials and coatings are well known in the art which may beincorporated onto the surface of the device or otherwise integrated intothe device. The radiopaque materials may be incorporated over the entiredevice or in discrete regions in any number of patterns to allow fordetection. In one embodiment, all or part of the expandable element mayinclude radiopaque materials. In some embodiments the distal tip of thedevice includes radiopaque materials.

As noted, the device may conveniently be configured as a catheter sizedfor passage through the lumen or over the body of an endoscope,laparoscope or other invasive scope device. The catheter may include anelongated shaft of circular cross-sectional shape having proximal anddistal ends; and an inflatable balloon disposed on the elongated shaft(FIG. 5). The balloon may be disposed at any point along the cathetershaft. In an exemplary embodiment, the balloon is disposed at the distalend of the shaft. In various embodiments, the balloon includes a wallhaving proximal and distal portions and having interior and exteriorsurfaces, the interior surface of the balloon wall being secured to theshaft in a fluid-tight manner. To facilitate inflation of the balloonthe shaft may include an inflation lumen in fluid communication with theballoon whereby fluid or gas can be infused and withdrawn to inflate andto deflate the balloon. Fluid or gas may be infused or withdrawn throughan accessory port in fluid communication with the inflation lumen whichmay be adapted to couple with a syringe, external pump, or the like.

A shown in FIGS. 5-7, the device of the present invention may beconfigured in a variety of ways to facilitate use of the device withother medical devices. In various embodiments, the device may include amain lumen that extends along the length of the elongated shaft. Thelumen may also be sized such that other medical devices may beintroduced within the main lumen. For example, an endoscope may beadvanced along the main lumen to allow for correct positioning of thedevice. In various embodiments, the elongated shaft of the device may beflexible or rigid.

With reference to FIG. 5, a particularly preferred deployment/retrievaldevice for use in the methods of the invention is configured as acatheter having an inflation balloon disposed at the distal end of thedevice. The device is configured such that it may be advanced though thelumen of an endoscope or overtube into a cavity of a subject. The devicemay include one or more opening in the elongated shaft to allow fordeployment of the microgrippers. In the embodiment depicted in FIG. 5, aplurality of openings are disposed proximal to the balloon. However, inother embodiments, the openings may be disposed at any point along theshaft of the device, for example, openings may be disposed through theballoon region or distal to the balloon.

In various embodiments, the microgrippers are deployed through acatheter through openings which may be disposed in any number ofconfigurations. The microgrippers are typically suspending in a smallvolume of fluid in a syringe, such as water. The syringe may then beattached to the catheter and the microdevices injected.

As shown in FIG. 5, the distal inflation balloon includes magnetsdisposed in a radially circumferential pattern around the balloon. Oneof skill in the art would understand that the magnets may be disposed inother pattern to achieve similar functioning of the device, such asbeing disposed as longitudinal stripes or being disposed over the entiredistal end or balloon of the device. In various embodiments, the magnetsmay be flexible or rigid.

With reference to FIG. 6, in one embodiment, the present inventionprovides a device configured as an overtube having an inflation balloondisposed at the distal end of the device. The device is configured suchthat it allows advancement of a second device along a lumen of thedevice. For example, an endoscope or catheter may be advanced along thelumen of the device facilitate placement of the device with the a tissuecavity of a subject. In one embodiment, an endoscope or catheter isadvanced along the lumen of the device before the device is place withinthe tissue cavity. The device is then advanced along the tissue cavityto a desired location at which point the device may be anchored byinflating the distal balloon.

One skilled in the art would understand that the device may include anynumber of balloons or other expandable elements disposed along theelongated shaft. For example, 1, 2, 3, 4 or more balloons or otherelements may be disposed along the shaft.

For clinical use, the present invention provides a method of tissuesampling. The method includes: a) contacting a plurality of tetherlessmicrotools with a tissue of a subject, each microtool having a firstconfiguration; b) allowing the plurality of tetherless microtools toalter from the first configuration to a second configuration while incontact with the tissue, the second configuration adapted such that eachmicrotool grasps the tissue at a discrete location; c) retrieving theplurality of tetherless microtools from the subject using a devicecomprising one or more magnets, wherein each microtool retains anindividual sample of the tissue upon retrieval, thereby performingtissue sampling. In various embodiments, the tetherless microtools aredeployed and/or retrieved from the subject using the device of thepresent invention.

For example, where the retrieval device of the invention is a catheterhaving an inflation balloon disposed at its distal end, the catheter maybe advanced along a tissue cavity along with an endoscope disposed inthe lumen of the catheter. Upon reaching a desired location, thecatheter may be anchored by inflating the balloon. The endoscope is thenremoved.

Microgrippers may be deployed through the catheter or otherwise beforeor after anchoring of the catheter. The device of the invention havingan elongated shaft including a distal balloon and main lumen extendingalong the length of the shaft is then passed over the anchored catheter.Once the distal tip of the device reaches the anchoring balloon of thecatheter, the anchoring balloon is deflated and the balloon of thedevice is inflated. Magnets disposed in the balloon of the devicecontact microgrippers within the body cavity. The device and catheterare then removed simultaneously from the body cavity.

Any number of microgrippers may be deployed within a body cavity. Invarious embodiments, 1 to 10, 1 to 50, 1 to 100, 1 to 250, 1 to 500, 1to 1000 or greater microgrippers are deployed and retrieved from a bodycavity. As such, one of skill in the art would understand that varyingamounts of tissue may be retrieved from the cavity which may be utilizedfor analysis.

The device and methodology of the invention allow for randomizedstatistical sampling of tissue. As such, in one aspect, the inventionprovides a method of obtaining a randomized statistical tissue sampleduring a diagnostic biopsy of a subject. The method includes a)introducing a plurality of tetherless microtools into a cavity of asubject; b) introducing a device comprising an expandable element havingone or more magnets into the cavity; c) anchoring the device via theexpandable element so as to facilitate contact between the expandableelement and the plurality of tetherless microtools; d) withdrawing thedevice so that the plurality of tetherless microtools collect on theexpandable element via magnetism and are removed from the subject,wherein each of the plurality of tetherless microtools comprise anindividual tissue sample from the cavity; and e) processing the samplesfor diagnostic biopsy.

As used herein, the term “sample” refers to any biological materialretrieved from a subject by a microgripper. For example, a sample can beany sample that includes a cell or other biological material that may beutilized for further analysis, including, for example, a tissue, abodily fluid, or a sample of an organ.

The term “subject” as used herein refers to any individual or patient towhich the subject methods are performed. Generally the subject is human,although as will be appreciated by those in the art, the subject may bean animal. Thus other animals, including mammals such as rodents(including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits,farm animals including cows, horses, goats, sheep, pigs, etc., andprimates (including monkeys, chimpanzees, orangutans and gorillas) areincluded within the definition of subject.

The methodology and device of the present invention may be deployed tobiopsy or obtain a sample of any area of the body. Generally,microgrippers are deployed to a body cavity, such as a hollow organ ofthe body, for example the gastrointestinal tract. However, microgrippersmay be deployed to any area of the body to obtain a sample. As such, theterm “body cavity” is intended to refer to internal surfaces and spacesof the body, as well as external surfaces of the body. In exemplaryembodiments, a sample is obtained from a mucosal or epithelial celllined surface, such as the pulmonary or respiratory tract, orgastrointestinal tract. In other exemplary embodiments, a sample isobtained from a duct or gland, such as a bile duct. In other exemplaryembodiments, a sample is obtained from an organ or tissue.

In various embodiments, the method of the invention further includesanalyzing the individual samples retrieved by the microtools. One ofskill in the art would appreciate that any number of analysis may beperformed using the samples to facilitate diagnosis or prognosis of acondition or disorder.

Accordingly, in another aspect, the present invention provides a methodof diagnosing a condition or disease in a subject. The method includes:a) obtaining a tissue sample from a subject via the method of theinvention; b) analyzing the tissue sample; and c) providing a diagnosisof the condition or disease based on the analysis.

In another aspect, the present invention provides a method of providinga prognosis for a condition or disease in a subject. The methodincludes: a) obtaining a tissue sample from a subject via the method ofthe invention; b) analyzing the tissue sample; and c) providing aprognosis of the condition or disease based on the analysis.

As such, the method of the present invention may be used, for example,to evaluate cancer patients and those at risk for cancer. In any of themethods of diagnosis or prognosis described herein, either the presenceor the absence of one or more indicators of cancer, such as, a cancercell, or of any other disorder, may be used to generate a diagnosis orprognosis.

Samples obtained from a subject may be used to investigate and identifyany number of conditions or disorders. As used herein, the terms“condition,” “disease,” or “disorder” are used to refer to a variety ofpathologies. For example, the term may include, but is not limited to,various cancers, immune pathologies, neurodegenerative diseases, and thelike. The term “cancer” as used herein, includes a variety of cancertypes which are well known in the art, including but not limited to,dysplasias, hyperplasias, solid tumors and hematopoietic cancers.Additional cancers may include, but are not limited to, the followingorgans or systems: brain, cardiac, lung, gastrointestinal, genitourinarytract, liver, bone, nervous system, gynecological, hematologic, skin,breast, and adrenal glands. Additional types of cancer cells includegliomas (Schwannoma, glioblastoma, astrocytoma), neuroblastoma,pheochromocytoma, paraganlioma, meningioma, adrenalcortical carcinoma,medulloblastoma, rhabdomyoscarcoma, kidney cancer, vascular cancer ofvarious types, osteoblastic osteocarcinoma, prostate cancer, ovariancancer, uterine leiomyomas, salivary gland cancer, choroid plexuscarcinoma, mammary cancer, pancreatic cancer, colon cancer, andmegakaryoblastic leukemia; and skin cancers including malignantmelanoma, basal cell carcinoma, squamous cell carcinoma, Karposi'ssarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma,keloids, sarcomas such as fibrosarcoma or hemangiosarcoma, and melanoma.

Once obtained, a sample is typically further processed and analyzed.Processing of the sample may include isolation of a biological componentof the sample, such as a protein, a nucleic acid molecule, or individualcell. In some embodiments, the sample is processed to isolate nucleicacids, such as DNA and RNA. In some embodiments, the sample is processedto isolate individual cells.

The term “nucleic acid molecule” is used broadly herein to mean asequence of deoxyribonucleotides or ribonucleotides that are linkedtogether by a phosphodiester bond. As such, the term “nucleic acidmolecule” is meant to include DNA and RNA, which can be single strandedor double stranded, as well as DNA/RNA hybrids. Furthermore, the term“nucleic acid molecule” as used herein includes naturally occurringnucleic acid molecules, which can be isolated from a cell, as well assynthetic molecules, which can be prepared, for example, by methods ofchemical synthesis or by enzymatic methods such as by the polymerasechain reaction (PCR), and, in various embodiments, can containnucleotide analogs or a backbone bond other than a phosphodiester bond.

The terms “polynucleotide” and “oligonucleotide” also are used herein torefer to nucleic acid molecules. Although no specific distinction fromeach other or from “nucleic acid molecule” is intended by the use ofthese terms, the term “polynucleotide” is used generally in reference toa nucleic acid molecule that encodes a polypeptide, or a peptide portionthereof, whereas the term “oligonucleotide” is used generally inreference to a nucleotide sequence useful as a probe, a PCR primer, anantisense molecule, or the like. Of course, it will be recognized thatan “oligonucleotide” also can encode a peptide. As such, the differentterms are used primarily for convenience of discussion.

In various embodiments, a sample may be analyzed in a variety of ways toidentify a condition or disorder. Typically, the analysis includeinvestigation of one or more nucleic acid molecules to diagnose adisease or condition, e.g., genetic analysis. In some embodiments, asample may be analyzed by performing image analysis of individual cellsto, for example, characterize cell type and cell morphology. Detectablemarkers, such as cell surface markers and nuclear markers, cell type,cell size, cell shape and the like may be analyzed using variousmicroscopy and imaging techniques known in the art.

As used herein, “marker” refers to any molecule that can be observed ordetected. For example, a marker can include, but is not limited to, anucleic acid, such as a transcript of a specific gene, a polypeptideproduct of a gene, a non-gene product polypeptide, a glycoprotein, acarbohydrate, a glycolipid, a lipid, a lipoprotein or a small molecule.

Any number of analyses may also be performed of the sample or componentthereof, to provide clinical assessment. For example, gene expressionanalysis and PCR techniques may be employed, such as gene chip analysisand multiplexing with primers specific for particular markers to obtaininformation such as the type of a tumor, metastatic state, and degree ofmalignancy. In some embodiments, expression of individual genesassociated with a disease or disorder is examined. In some embodiments,expression of cell surface markers may be analyzed. As used herein,“expression” refers to the production of a material or substance as wellas the level or amount of production of a material or substance. Thus,determining the expression of a specific marker refers to detectingeither the relative or absolute amount of the marker that is expressedor simply detecting the presence or absence of the marker.

Additionally, cell size, DNA or RNA analysis, proteome analysis, ormetabolome analysis may be performed as a means of assessing additionalinformation regarding characterization of a disease or disorder. Invarious aspects, analysis may include antibodies directed to or PCRmultiplexing using primers specific for one or more of markers. Examplesof well known cancer markers include: EGFR, HER2, ERCC1, CXCR4, EpCAM,E-Cadherin, Mucin-1, Cytokeratin, PSA, PSMA, RRM1, Androgen Receptor,Estrogen Receptor, Progesterone Receptor, IGF1, cMET, EML4, andLeukocyte Associated Receptor (LAR).

Analysis may also include performing methylome analysis or detecting themethylation status of an isolated nucleic acid molecule. In variousembodiments, the determining of methylation status is performed by oneor more techniques selected from a nucleic acid amplification,polymerase chain reaction (PCR), methylation specific PCR, bisulfitepyrosequenceing, single-strand conformation polymorphism (SSCP)analysis, restriction analysis, microarray technology, and proteomics.Analysis of methylation can be performed by bisulfite genomicsequencing. Bisulfite treatment modifies DNA converting unmethylated,but not methylated, cytosines to uracil. Bisulfite treatment can becarried out using the METHYLEASY™ bisulfite modification kit (HumanGenetic Signatures). Other methods are known in the art for determiningmethylation status, including, array-based methylation analysis,Southern blot analysis, molecular beacon technology, Taqman™ technology,methyl light, Methyl Heavy, or SNuPE (single nucleotide primerextension). The degree of methylation in a nucleic acid molecule mayalso be measured by fluorescent in situ hybridization (FISH).

With regard to cancer, analysis allows for meaningful characterizationuseful in assessing diseases prognosis and in monitoring therapeuticefficacy for early detection of treatment failure that may lead todisease relapse. In addition, analysis enables the detection of earlyrelapse in presymptomatic patients who have completed a course oftherapy. Thus, enumeration and characterization of specific cells of asample provides methods to stratify patients for baselinecharacteristics that predict initial risk and subsequent risk based uponresponse to therapy.

In various aspects, samples may be obtained and analyzed over aparticular time course in various intervals to assess a subject'sprogression and pathology. For example, analysis may be performed atregular intervals such as one day, two days, three days, one week, twoweeks, one month, two months, three months, six months, or one year.

Analysis may provide data sufficient to make determinations ofresponsiveness of a subject to a particular therapeutic regime, or fordetermining the effectiveness of a candidate agent in the treatment of adisease or disorder, such as cancer. For example, once a drug treatmentis administered to a patient, it is possible to determine the efficacyof the drug treatment using the method of the invention. For example, asample taken from the patient before the drug treatment, as well as oneor more cellular samples taken from the patient concurrently with orsubsequent to the drug treatment, may be isolated and processed usingthe method of the invention. By comparing the results of the analysis ofeach processed sample, one may determine the efficacy of the drugtreatment or the responsiveness of the patient to the agent. In thismanner, early identification may be made of failed compounds or earlyvalidation may be made of promising compounds.

Analysis of a sample during a clinical trial will provide information onwhether the patient is responding or not responding to an experimentaldrug. This information is an early indicator of the drug's effectivenessand may be used by the investigators as a secondary endpoint in theclinical trial.

The following examples are provided to further illustrate the advantagesand features of the present invention, but are not intended to limit thescope of the invention. While they are typical of those that might beused, other procedures, methodologies, or techniques known to thoseskilled in the art may alternatively be used.

Example 1 Tissue Sampling of Pig Colon

In a significant step towards enabling non-invasive surgery, thisexample describes sampling of gastrointestinal tissue usingsub-millimeter sized tetherless microtools, in the form ofmicrogrippers. The microgrippers were fabricated using conventionalmultilayer microfabrication and were composed of rigid segmentsconnected to pre-stressed metallic bilayer hinges covered by athermo-sensitive polymer trigger. On exposure to body temperature, theopen microgrippers close spontaneously, typically within five minutes.This example shows ex-vivo proof-of-concept tissue sampling by deployingthe microgrippers through a catheter. Subsequent retrieval of thegrippers with a magnetic probe enabled tissue acquisition for viablemolecular diagnostics. Microgrippers were used to retrieve tissueendoscopically, from which RNA and DNA were extracted. Polymerase-chainreaction (PCR) was also performed for several genes on the extractedgenetic material.

Microgrippers were modeled as biological appendages, such as claws, bydesigning flexible joints between rigid regions, as described in Leonget al. (Proc Natl Acad Sci USA 106:703-8 (2009)). Due to their smallsub-mm size (˜0.98 mm from tip to tip when completely open), it waspossible to deploy hundreds of them. The rigid regions of the grippersare composed mostly of nickel, a ferromagnetic material, so that thegrippers can be guided with an external magnetic field from afar. Also,the nickel parts were covered by gold to render them bio-inert. FIG. 1shows the schematic representation of the grippers and their operationused in this example.

The following experimental methods and protocols were utilized.

Microgrippers. The microgrippers were fabricated on silicon substratesutilizing standard photolithography techniques and released from thesubstrate prior to the endoscopic experiments as described in Leong etal. (Proc Natl Acad Sci USA 106:703-8 (2009)). After the grippers werereleased from the substrate, they were rinsed several times in DI waterand kept at ˜0° C. till their deployment to the colon.

Animal Tissue. Pig colons were obtained from a local slaughterhouseminutes after the animals were butchered. The pig colons wereimmediately placed and kept on ice until the next day when theendoscopic experiments were performed. The resection piece included anintact colon, from anus to cecum. To facilitate endoscopic visualinspection of the pig colon, the colon was tied at the cecum with aplastic band, so that air insufflation would result in distension of thecolon and adequate mucosal inspection.

Endoscopic Equipment. For the endoscopic experiments a standard Pentax™EG-3840T double channel (therapeutic) endoscope was used. A singlechannel endoscope can also be used in a similar fashion with theexception that the catheter used to deploy the grippers needs to beremoved prior to re-using the same port for the magnetic catheter toretrieve the microgrippers.

Microgripper Retrieval and Analysis. A grade N52, Neodymium magnet (K&JMagnetics Inc, Jamison, Pa.) was used to build a magnetic catheter forendoscopic retrieval of grippers. A group of retrieved grippers werestained with trypan blue (Sigma-Aldrich, St. Louis, Mo.) to image thecellular media under the optical microscope. Another group of gripperswere quickly submerged in Trizol (for RNA extraction) or Viagen solution(for DNA extraction) immediately after the retrieval.

DNA and RNA Extraction. The RNA was extracted by using TRIzol™ reagent(Invitrogen, Carlsbad, Calif.). RNA specimens were stored at −80° C.prior to analysis. Reverse transcription was performed by using the kitfrom Fermentas (Catalog number K1612) (Fermentas Inc, Glen Burnie, Md.).DNA was extracted with the Viagen solution (Catalog number 102-T)(Viagen Biotech, Los Angeles, Calif.), by following the manufacturer'sprotocol.

Results

Swine Model for Ex-vivo Colonoscopy. To evaluate the feasibility ofstatistical tissue sampling from hollow gastrointestinal organs usingmicrogrippers, ex-vivo colonoscopies were performed on pig colon, whichwas chosen because of its similarities to human colon in terms of sizeand endoscopic appearance. A human therapeutic (double channel) upperendoscope was used for these experiments. The endoscope was insertedthrough the anus and advanced under endoscopic guidance to the cecum andthen withdrawn while the mucosa was inspected (FIG. 8a ). Except formucosal pallor, which was expected given the lack of blood supply, thecolonic mucosa appeared remarkably similar to human colonic mucosa (FIG.8b ).

Endoscopic Deployment and Intraluminal Activation of the Microgrippers.The hinge layer of the microgrippers is thermo-sensitive and whenexposed to the transition temperature, (37° C.), the hinges bend and themicrogrippers close. After being subjected to this temperature, it takesabout 5 minutes for the grippers to completely close and grip proximaltissue. Prior to the procedure, the open grippers were kept in coldwater (−0° C.) to prevent premature closing. To mimic the normal humanbody temperature, the colon was placed in a water bath kept at 37° C. Acatheter was inserted through one of the channels of the endoscope.

In the first set of experiments, approximately 30 microgripperssuspended in sterile water were aspirated into the catheter and thendeployed on an area approximately 4 cm² in size. FIG. 9a shows anendoscopic image of the deployed grippers on colonic mucosa. The numberof deployed microgrippers was varied later to test for the ability tospread the grippers for a wide sampling of the colonic mucosa. A uniformspread of the microgrippers was achieved when the endoscope wascontinuously whirled during the deployment of large number of grippers(approximately 100) as seen in FIG. 9b . The number of deployed grippersis important to improve the statistical efficacy of sampling.

When grippers were deployed in the colon, soon after mucosal contact,they were activated by the colonic temperature (37° C.) and closed. Theclosure of the grippers was verified endoscopically. Most grippersclosed onto the colonic mucosa (FIG. 9c ) while very few grippers closedtowards the colon cavity.

Magnetic Retrieval of the Grippers. A specially designed catheterfeaturing a magnetic tip was used to retrieve the microgrippers. Thecatheter was inserted through the second channel of the endoscope andused to collect the grippers; FIG. 9d shows the retrieval of thegrippers by magnetic catheter. The vast majority of grippers weresuccessfully captured with the magnetic catheter and the rest weresuccessfully suctioned with the endoscope into a trap bottle.

A group of retrieved grippers were imaged with optical microscopy toverify that they obtained tissue from the colonic mucosa, FIG. 10a .Next, the grippers and associated tissue were separated from themagnetic tip and stained with trypan blue, a tissue stain. FIG. 10bshows that the blue stained cellular material is attached to thegrippers confirming that the retrieved grippers returned with cellularmaterial.

Genetic Analyses of RNA and DNA of the Tissue Obtained Through theGrippers. A second group of retrieved grippers was used to extract RNAand DNA from the obtained tissue. The concentration of RNA was measured,on average 122 ng/μL and the concentration of DNA was measured, onaverage, 134 ng/μL. The RNA was reverse transcribed, and thecomplementary DNA (cDNA) was utilized as template for PCR. Primersdesigned for pig Beta-actin, Cyclophilin-A (CPA) and Interleukin-6(IL-6) were employed, because of the relative high abundance of thesetranscripts. As FIG. 10c demonstrates, cDNA amplification produced bandsof expected size. Similarly, DNA primers designed for pig DNA Kalirin(KALRN), Mucin 4 (MUC4) and Integrin beta 5 (ITGB5) were employed, dueto the relative high abundance of these genes. Subsequently, standardPCR followed by gel electrophoresis were performed. FIG. 10ddemonstrates that all 3 genes tested were successfully amplified andthat the amplified DNA had the expected size.

In cases where the macroscopic appearance of the mucosa is unrevealing,random biopsies are required. In these cases, broad mucosal coverage isdesired. In addition, making a diagnosis of a premalignant or malignantcondition is oftentimes more important than the precise location of theabnormality. Examples include diagnosing dysplasia in ulcerativecolitis, microscopic/collagenous colitis or Helicobacter Pyloriinfection. This example describes an innovative endoscopic biopsyingtechnique that employs tetherless microgrippers. These microgrippers canbe deployed, activated and retrieved endoscopically, as demonstrated bythe experiments on pig colon.

The device and methodology of the present invention differssignificantly from the traditional biopsy forceps. Due to miniaturizedsize of the microgrippers and their deployment method, e.g., sprayingthe grippers through the catheter, it is possible to achieve expansiverandomized biopsy sites and consequently better mucosal coverage. Inaddition, the microgrippers, due to their minute size, likely result inless mucosal damage than the traditional biopsying forceps potentiallyresulting in fewer endoscopic side effects.

Due to the sub-millimeter size of the grippers, the amount of tissueretrieved by each gripper is significantly less than the tissue obtainedby traditional biopsy forceps. However, the amount of tissue gathered bytraditional forceps from each biopsy sites may be unnecessary forgenetic or epigenetic diagnoses. As show in this example, the tissueretrieved by the grippers is of sufficient quality and quantity to allowDNA as well as RNA extraction.

In an era of major molecular biology advances on genome sequencing aswell as on mRNA, miRNA and epigenetic studies, an ever increasing numberof gene alterations can be identified. These alterations, eitherindividually, or in combination, can be utilized to diagnose diseases.When several mucosal conditions are not readily detectable bytraditional endoscopic inspection, a protocol of randomized biopsy isnecessary. In these cases, the best mucosal coverage obtained withminimal damage to the rest of the mucosa is highly preferred. Thisexample demonstrated the feasibility of using tetherless thermo-actuatedmicrogrippers for the randomized endoscopic tissue sampling.

Example 2 In-Vivo Bile Duct Tissue Sampling Through EndoscopicRetrograde Cholangiopancreatography (ERCP)

This example describes sampling of bile duct tissue through ERCP.

The microgrippers were fabricated on silicon substrates utilizingstandard photolithography techniques and released from the substrateprior to the endoscopic experiments as described in Leong et al. (ProcNatl Acad Sci USA 106:703-8 (2009)) and discussed in Example 1.

Microgrippers were deployed into the bile duct through an ERCP catheterand retrieved via a device of the present invention configured as amagnetic catheter. FIG. 11 shows introduction of the catheter. FIG. 12ashows retrieved microgrippers on the magnet of the catheter as well asretrieved microgrippers detached from the magnet and stained with trypanblue.

After removal of the microgrippers, the liver of the pig was removed toassess the retrieval rate of microgrippers. The liver was image thoughmagnetic resonance. Only 2 grippers were found to remain in the liver,whereas more than 40 were successfully retrieved. As such the retrievalrate observed was greater than 95%.

Example 3 Enzymatically Actuated Microgrippers

This example describes sampling using enzymatically actuatedmicrodevices.

Multilayer grippers with hinges composed of either gelatin, apolypeptide, or carboxymethylcellulose (CMC), a polysaccharide weregenerated as disclosed in Bassik et al. (J Am Chem Soc 132:16314-7(2010)). These hinges contained pre-stressed and structural metal films,were patterned using photolithography and combined with rigid segmentsto create a gripper. These tools closed and re-opened when exposed toproteases and glucosidases respectively. They were approximately 1.1 mmin diameter when open and approximately 600 μm in diameter when closed(FIG. 13). The fabrication process was highly parallel and approximately1600 grippers could be fabricated simultaneously on a three inchdiameter wafer.

Grippers were designed with alternating rigid segments and flexiblehinges. Rigid segments remained flat during the entire cycle of closingand re-opening, providing the mechanical strength required for securegripping. Flexible hinges were initially flat, and curved only onexposure to the appropriate enzyme. Closing and re-opening was achievedusing a gripper design with two kinds of hinges which bent with eitherconcave or convex curvatures. The concept behind this two-stageactuation is as follows: on the appropriate trigger, all hinges coveredwith one biopolymer actuate, while the entire second set of hingesremain flat, causing the gripper to close. As the modulus of the secondbiopolymer is reduced, all the second hinges also bend, but in theopposite direction, thus re-opening the gripper (FIG. 13b ).

The grippers were microfabricated in two dimensions (2D) on a siliconwafer using conventional photolithography techniques and weresubsequently released from the substrate. The first step involveddepositing a sacrificial copper (Cu) layer on the wafer by thermalevaporation. Next, flexible and rigid components consisting of chromium(Cr), nickel (Ni) and gold (Au) were deposited and patterned by lift-offmetallization and electrodeposition. Ni, which is ferromagnetic, wasincorporated to allow for remote magnetic manipulation. The gripper wasdesigned such that only Cr and Au surfaces were exposed to render itbio-inert. Aqueous biopolymer solutions were dispensed onto the featuresand patterned by exposure to ultraviolet (UV) light through a quartzphotomask. The uncrosslinked biopolymer was washed away, and thesacrificial layer was dissolved to release untethered grippers.

Commercially processed derivatives were chosen of natural biopolymers toallow for aqueous handling: gelatin, derived from collagen, and CMC,from cellulose. The two biopolymers are targeted by different familiesof enzymes—without overlapping activity—so that each set can be actuatedselectively. Additionally, it is known that gelatin is degraded byenzymes which occur in disease states, such as proteases in cancer. Thisoffers the possibility for autonomous actuation in response to a diseasemarker. The other biopolymer, CMC, degrades on exposure to non-mammalianenzymes that do not interact with animal tissue.

Both raw biopolymers were synthetically modified by chemical grafting ofmethacrylate groups to the polymer backbone. This modification enabledcross linking under UV light in the presence of molecular crosslinkersand free-radical photoinitiators. The highlight of this process is thatthe crosslinked material retained the necessary accessible monomergroups to allow for enzymatic recognition and cleavage.

The biopolymers were patterned sequentially atop the metal layers, whileunderlying multilayer hinges were constructed by layering thin filmswith specific levels of tension. The bending angles of the hinges weredesigned using a multilayer mechanics model. The magnitude of the straindifferential across the thickness of this multilayer stack leads to aspecific bending angle at equilibrium. Controlled bending of themultilayer hinge was achieved by altering the mechanical properties ofthe biopolymer. A relatively stiff crosslinked biopolymer on top of apre-stressed bilayer stack arrested its bending, causing it to remainflat. Removal or softening of this biopolymer allowed the hinge to bend(FIG. 14).

Using the model, it was determined that for a typical gel modulus of 10⁴Pa, a 150 μm thick patterned gel would be sufficient to ensure a flatstate, which was verified experimentally. In order to create aminiaturized integrated tool we used a computer simulation to modelserial linkages of rigid segments and various hinge types as a 2D crosssection, so that the folding state of any tool could be visualized insilico before experiments. Grippers were first simulated with a singleset of hinges that simply closed on actuation. Using this model, it wasobserved that a reduction in modulus to 100 Pa would cause the gripperto close, and observed a similar actuation profile in experiments (FIG.2g ).

Grippers were then modeled with two sets of hinges that closed andre-opened. Closing occurred when the modulus of the first biopolymer(CMC) was reduced to 100 Pa while the second biopolymer (gelatin)remained stiff (10⁴ Pa) (FIG. 14a-e ). Reopening occurred when themodulus of gelatin was reduced in turn. During the actuation of thegelatin-triggered hinge, the modulus of CMC remained low and was notaffected.

Selectivity of actuation was studied using grippers with either agelatin or a CMC trigger. Several enzymes were screened includingproteases from animal pancreatic origin (trypsin), plant origin(papain), and bacterial origin (collagenase), which are specific to thepolypeptide (gelatin) grippers, and carbohydrate degrading enzymes fromfungal origin (cellulase) that are specific to the polysaccharide (CMC)trigger. A commercial mixture of many carbohydrases used for plant cellwall lysis (Viscozyme) were tested, as well as phosphate buffered saline(PBS) and cell culture media with serum. For these experiments, specialcare was taken to ensure that all grippers were fabricatedsimultaneously on a single wafer and differed only in the application ofgelatin or CMC hinge triggers as a final step.

In FIG. 2g , we show the response of the tools to different hydrolyticenzymes. Using optical microscopy, the diameter (D) of grippers exposedto different enzymes was recorded over time. The parameter D/Dmax, wasutilized which allowed quantification of closing and re-opening invarious enzymes, and plot average data for 5 grippers. Open grippers atmaximum spread have a defined edge-to-edge ratio D/Dmax of 1, and inthis specific design, completely closed grippers have a theoreticalminimum ratio of 0.355. As an example, it was experimentally observedthat polypeptide grippers in trypsin fold to 90% of maximum(D/Dmax=0.42) in approximately 10 minutes.

It was observed that none of the grippers closed in the presence of PBSor mammalian cell media over 60 days. Upon addition of proteases such astrypsin, collagenase, or papain, sufficient degradation of the gelatinresults in closing within 30 minutes. Proteases did not degrade thepolysaccharide layers for a minimum of 30 days. After several weeks,some specificity was lost, as polypeptide grippers closed in cellulasein one week, and several polysaccharide grippers closed in papain at onemonth. This observation is attributed to the use of crude enzymeextracts from plant and bacterial sources that likely mixedprotease/polysaccharidase activity.

Sensitivity of the grippers to decreasing concentrations of enzymeactivity was also examined. Serial dilutions of collagenase were takencovering three orders of magnitude (5400 Units/mL to 21 Units/mL) andincubated UV sterilized gelatin grippers in the solutions. While thegrippers in the highest concentrations closed in minutes, they tookseveral weeks to close in the lowest enzyme concentrations. Similarly,CMC grippers exposed to a 2250 Unit/mL cellulase solution closed inunder 5 minutes as compared to approximately 18 hours in a 27 Unit/mLdilution. Decreased enzyme activity is expected to increase closing timeas both bond cleavage and diffusion will be reduced. However, theability to actuate in varying enzyme concentrations suggests possibleactivity in vivo.

The gripper actuation was sensitive to the appropriate enzyme class forrapid actuation. This allowed a time window for differential actuationof orthogonal enzymes. Actuation of hinges in series was demonstrated byplacing the grippers in solutions of papain and then cellulase, or viceversa. Orthogonal actuation of specific grippers was observed in onlythe corresponding enzymes.

The enzymatically triggered grippers were used to demonstrate medicallyrelevant tasks. It is difficult to reach closed lumina in the body, suchas the biliary tree, with tethered tools. One alternative is tomanipulate untethered devices using magnetic forces, also permittingvisualization via magnetic resonance imaging. CMC-gelatin grippers thatcould be closed and reopened were used to securely grip a 700 μmalginate bead. Closing was actuated using a cellulase trigger. Thegripper with the bead securely in its grasp was moved using a magneticstylus and subsequently release the bead using a collagenase trigger.This demonstration highlights possible applicability in pick-and-placeoperations and on-demand drug delivery.

Avian liver tissue was biopsied from a model organ (cast from acrylicresin) with size scales approximately that of an adult human. For thisexperiment we utilized CMC grippers that closed in response tocellulase. After placing the grippers in the duodenum, they wereremotely piloted them through the ampulla of Vater, through the commonbile duct (5 mm diameter lumen), and into the liver. Cellulase solutionwas then added via syringe and the gripper closed around the tissue.Magnetic manipulation was used to extract the gripper and excisedtissue, which was then stained (data not shown).

Additionally, the ability of these tools to retrieve cells for furtherdiagnostic analysis was tested. A line of normal, SV-40 transformed,bile duct cells (H69) and a line of cholangiocarcinoma cells (HuCCTl)were cultured. After collecting the cells in a pellet CMC triggeredgrippers were introduced and closed them with cellulase (data notshown). The force exerted by an external magnet used to move thegrippers was sufficient to hold up a large (9 mm) cell clump with eightgrippers. Both cell types were analyzed via gel to confirm the presenceof RNA (data not shown). This technique suggests that collection ofclinically relevant data from gripper biopsy is feasible.

In conclusion, grippers triggered by specific enzyme substrateinteractions have been demonstrated. These hybrid metal/polymer toolsallow creation of miniaturized devices and materials that respondautonomously to specific biochemicals and disease markers. For example,by matching the biopolymer to proteolytic enzymes that are naturallysecreted from cancer cells it should be possible to facilitate a toolthat responds only to cancerous environments. It should also be possibleto delay actuation by crosslinking protease inhibitors into thebiopolymer or accelerate it using protease zymogens such as trypsinogen.Such methodology may be extended to other enzyme-biopolymer pairs suchas the degradation of DNA based biopolymers using nucleases. Inprinciple, the process is also compatible with nanoscale patterningtechniques such as electron beam or direct write techniques whichsuggests the possibility of further miniaturization.

Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

What is claimed is:
 1. A system for obtaining a tissue sample from asubject, the system comprising: a) a device comprising: i) an elongatedtubular shaft having proximal and distal ends; and ii) an inflatableballoon disposed on the distal end of the shaft, the inflatable balloonhaving one or more magnets disposed thereon and configured for retrievalor deployment of a tetherless microtool, wherein the shaft has one ormore openings disposed proximal to the balloon for deployment of thetetherless microtool from a lumen of the shaft; and b) a tetherlessmicrotool magnetically detachable to the one or more magnets.
 2. Thesystem of claim 1, wherein the device comprises a plurality of magnets.3. The system of claim 1, wherein the balloon comprises a wall havingproximal and distal portions and having interior and exterior surfaces,the interior surface of the balloon wall being secured to the shaft in afluid-tight manner.
 4. The system of claim 3, wherein the shaft furthercomprises an inflation lumen in fluid communication with the balloonwhereby fluid or gas can be infused and withdrawn to inflate and todeflate the balloon.
 5. The system of claim 1, wherein the device isconfigured as a catheter.
 6. The system of claim 1, wherein the devicefurther comprises a radiopaque material.
 7. The system of claim 1,wherein the tetherless microtool alters configuration in response to astimulus from a first configuration to a second configuration, thesecond configuration being capable of grasping tissue.
 8. The system ofclaim 1, wherein the tetherless microtools have a major dimension lessthan about 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mm.